One of the most important mediators of host resistance to a vast number of pathogens is the cytokine Interferon gamma (IFN?). During infection, IFN? is released from specialized immune cells and induces multiple antimicrobial response pathways in most mammalian cell types. An important component of the IFN? response is the induction of cell-autonomous resistance towards bacterial pathogens residing and replicating within vacuoles, including Mycobacterium tuberculosis and Legionella pneumophila. To exert their antimicrobial activities, numerous IFN? -induced host response proteins like Nitric oxide synthase 2 (Nos2), Immunity Related GTPases (Irg) and Guanylate binding proteins (Gbp) translocate to pathogen-containing vacuoles (PCVs). The principles that underlie the ability of the host cell to recognize PCVs and target IFN? -induced host proteins to them are currently not well understood. The goal of this research proposal is to fill this gap in our knowledge by defining the molecular players that direct the localization of IFN? -induced antimicrobial proteins to PCVs. Towards this goal, we are taking two complementary, genomic approaches that aim to identify host factors that either facilitate the translocation of IFN? -induced proteins to a Legionella-containing vacuole (LCV) or translocate to LCVs directly.
In Specific Aim 1, we will use gain- and loss-of-function screening approaches to identify host factors required for IFN? -mediated restriction of L. pneumophila replication inside macrophages. Host factors critical for providing resistance to L. pneumophila replication inside IFN? -activated macrophages will be subjected to mechanistic studies to further characterize their role in targeting IFN? - induced proteins to PCVs.
In Aim 2, we will use an epitope-tagged kinome expression library to directly identify kinases localizing to PCVs in IFN? -stimulated cells using a high-content imaging approach. This second approach will potentially allow us to identify functionally redundant host factors, as well as capture the dynamic sequence of events involved in the host response. Identification of these targeting pathways is a first and critical step towars understanding how host cells recognize PCVs as 'non-self'and potentially dangerous vesicular structures and, conversely, how host-adapted pathogens can evade recognition. This knowledge will open up avenues for the development of novel therapeutics that aim to boost the inherent ability of the host organism to detect and eliminate intracellular bacterial pathogens.
This proposal aims to deploy new (genomics-based) approaches to identify the key host factors responsible for the delivery of antimicrobial proteins to vacuoles that contain pathogens such as the bacterium Legionella pneumophila. Host factors identified in these studies are likely to play an important role in host resistance to many pathogens, including Mycobacterium tuberculosis and Salmonella enterica, and may serve as drug targets for novel host-based antimicrobial therapies.
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